HLA variation is a crucial determinant of transplant rejection, susceptibility to a large number of infectious and autoimmune diseases, and cancer. The limiting factor in large-scale genetic analysis of, for example, transplant populations has been methodologic and directly involves the technical ability to accurately define the alleles of highly polymorphic HLA genes in a cost-effective and efficient manner. Although recent progress in the development of traditional probe hybridization, sequencing and array-based methods has allowed alleles to be determined with accuracy, large-scale efforts in genetic analysis of transplant populations are hampered by the cost and inefficiency of available methods.
In particular, current array-based methods are inefficient, because resolution of ambiguities is not guaranteed, and multiple arrays are needed to conduct a complete analysis of all HLA genes. Exemplary limitations of currently available HLA arrays include for example, that they are suitable for low-to-medium density genotyping but not for high-density genotyping; they do not have a complete set of probes (i.e. capture oligonucleotides that recognize target HLA encoding nucleic acids), but rather have only a selection of “informative probes”, thereby making deconvolution of ambiguities difficult or impossible; they do not cover all currently known HLA alleles from each group; and they do not encode a complete set of classical and non-classical HLA loci. Current methodologies, e.g., microarrays, are typically limited to identifying one or several classical HLA molecules (e.g., HLA-A, HLA-B or HLA-C) per assay, and cannot simultaneously determine the haplotype of all classical MHC molecules. Nor can current methodologies simultaneously determine the haplotype of non-classical HLA molecules or accessory molecules (e.g., those important in antigen processing and peptide loading on HLA molecules) in the same assay, and thus do not efficiently provide a complete picture of an individual's HLA tissue type, as needed, e.g., to understand the linkage between certain tissue types and disease susceptibility, and for determining donor/recipient compatibility in tissue transplant.
There therefore exists a need in the art for new and improved methods for not only the identification of all alleles of any HLA molecule, but the ability to identify all alleles of all HLA molecules known at the time of identification, as well as certain other polymorphic molecules, in a single assay. This invention addresses these and other needs as described in detail below.